Diseases caused by positive-sense single-stranded RNA viruses, including poliovirus, rhinovirus, hepatitis C virus, and SARS, are critical health issues worldwide. Progression of disease requires replication of the virus genome by oligomeric complexes assembled on virally-induced membrane structures in the host cell. Design of drugs that interfere with protein-protein interactions has made considerable progress in recent years, making disruption of the viral replication complex an attractive target for antiviral intervention. This proposal focuses on poliovirus (i) in order to use the extensive knowledge of the structural and functional properties of poliovirus as a basis for detailed characterization of these protein-protein interactions, and (ii) to address the need for new antiviral strategies against poliovirus that a 2007 National Academy of Sciences panel asserted would significantly strengthen the eradication effort, and prevent its threat as a bioweapon against an unvaccinated population. Assemblies of poliovirus RNA polymerase, the functional centerpiece of the replication complex, will be analyzed in terms of the intermolecular interactions responsible for their stability and interpreted on the basis of high resolution crystallographic data and extensive mutational and biochemical data. As the field of structural electron microscopy advances toward routine sub-nanometer resolution, it is biological questions such as those asked here that continue to motivate technical innovations. In Specific Aim 1, methods for the routine use of cryo-electron microscopy to solve structures of nanocrystals as small as 10 unit cells on a side will be developed and used to analyze the structure of nanocrystals of RNA polymerase and characterize the intermolecular interactions therein. This will be of value far beyond the proposed studies of polymerase, enabling structural analyses of the 'shower' of protein nanocrystals that often appear in crystallization trays, that until now have been discarded as failed experiments, and that recently have been attracting greatly increased interest. In Specific Aim 2, a mechanism for stabilizing polymerase-RNA complexes and supporting RNA replication via a positively charged channel across the polymerase interface will be tested by structural and functional analyses of complexes comprising wild-type and mutant polymerases. In Specific Aim 3 transfection of viral replication proteins into mammalian cells will be used to (i) define th minimum viral component required for the membrane reorganization of cellular membrane into double-bilayers, and (ii) characterize the protein-protein interactions that stabilize polymerase-containing oligomers, as these interfaces represent sites of vulnerability for disruption of viral replication and for development as potential drug targets.